There has been considerable interest in the synthesis and testing of hydroxy-substituted benzohydroxamic acids, particularly as antimicrobial or antitumor agents. The leading research group in this area had been headed by Glen R. Gale. In 1966, Gale presented a paper in Proc. Soc. Exptl. Biol. Med., 122, 1236 (1966) on the selective inhibition of DNA synthesis by salicylhydroxamic acid. He reported that cells which had been preincubated with salicylhydroxamic acid for up to two hours showed only a slight depression of RNA and protein synthesis. Gale and Hynes reported in J. Med. Chem., 11, 191 (1968) on a similar activity of other arylhydroxamic acids including 3-hydroxy, 4-hydroxy, 2,6-dihydroxy, 2,3-dihydroxy, 3,5-diamino, 4-amino and 3-aminobenzohydroxamic acids. The compounds were tested for their ability to inhibit DNA synthesis in Ehrlich ascites tumor cells in vitro. The authors confirmed the selective inhibition of DNA synthesis by salicylhydroxamic acid. Benzohydroxamic acid was found to inhibit RNA synthesis but the corresponding benzamide was found to be totally inactive. 3,5-Diaminobenzohydroxamic acid and 4-aminobenzohydroxamic acid were said to display relative selectivity against DNA synthesis, but only after exposure of the cells to the agent for 1-2 hours. 2,3-Dihydroxybenzohydroxamic acid was said to show a marked selectivity against DNA synthesis with a greater inhibition than that produced by salicylhydroxamic acid. 3-Aminobenzohydroxamic acid was reported as completely inhibiting DNA synthesis but accompanied by 50 percent depression of RNA and protein synthesis.
The authors concluded as follows: "[t]he most active and selective compounds were, thus, those with hydroxyl substituents in the 2and 2,3-positions on the aryl ring, while addition of the same group to the 4-position yielded a virtually inactive compound. Relatively selective but less active compounds were those with amino groups in the 4 and 3,5-positions."
Howle and Gale publishing in Proc. Soc. Exptl. Biol. Med., 131, 697 (1969) [same as Chemical Abstracts 71, 57006b (1969)] describe the effects of certain hydroxamic acids on bacterial and plant L-glutamate 1-carboxylase. A few amino and hydroxy-substituted benzoylhydroxamic acids were studied. Among the new compounds reported was 2,3,4-trihydroxybenzoylhydroxamic acid.
Gale, Hynes and Smith publishing in J. Med. Chem., 13, 571 (1970) described the synthesis of additional arylhydroxamic acids which inhibited the synthesis of DNA in Ehrlich ascites tumor cells. The authors commented on work performed by the National Cancer Institute, N. Greenberg--Cancer Chemotherapy National Service Center, stating that 4-hydroxybenzoylhydroxamic acid possessed significant antitumor activity in vivo against L1210 leukemia. The sole new compound prepared disclosed was 2,5-dihydroxybenzoylhydroxamic acid. The most active compound reported was 3,5-diisopropylsalicylhydroxamic acid, with another interesting structure being 4-nitrobenzoylhydroxamic acid. The 2,5-dihydroxybenzoylhydroxamic acid derivative was apparently of little interest. The authors concluded that a majority of their derivatives which were active in vitro were substituted in the 4-position in relation to the hydroxamic acid group.
The most recent paper of interest by Gale and co-workers appeared in Biochemical Pharmacology, 20, 2677 (1971) [same as Chemical Abstracts, 76 21107z (1972)]. This paper reported the results of testing 20 arylhydroxamic acids as potential antimitogenic agents. Among the compounds tested were the following benzoylhydroxamic acid derivatives: 3-hydroxy, 4-hydroxy, 4-amino, 2,3-dihydroxy, 2,4-dihydroxy, 2,5-dihydroxy, 2,6-dihydroxy, 3,5-diamino. The most active compounds tested were 4-hydroxybenzoylhydroxamic acid and the corresponding 2,3-dihydroxy compound. 4-Aminobenzoylhydroxamic acid was found to be inactive as was the 2-hydroxy-4-aminobenzoyl compound. All compounds were compared with hydroxyurea, which compound acts on susceptible cells by inhibiting DNA synthesis through suppression of the activity of ribonucleoside diphosphate reductase. Although Gale and coworkers never tested the effect of their compounds on ribonucleotide reductase, it was the conclusion of the authors that 2,3-dihydroxybenzoylhydroxamic acid probably acted by a different mechanism. The authors also concluded that there was fundamental difference in action between the 4-hydroxy and the 2,3-dihydroxy benzohydroxamic acids.
Other investigators have also been interested in the properties of hydroxy-substituted benzoic acids and benzamides. Kreuchunas, U.S. Pat. No. 2,849,480, discloses a number of derivatives of 2,3,6-trihydroxybenzoic acid such as the amide, the N-methylamide, the N,N-dimethylamide, etc. Utility of the compounds is said to be in the treatment of rheumatic fever. Chemical Abstracts, 81, 120190f (1974) discloses the stabilization of aromatic amines with esters or amides of gallic acid (3,4,5-trihydroxybenzoic acid). The primary amide and the N-methylamide are specifically disclosed. Chemical Abstracts, 85, 94115w (1978) discloses a group of 2,6-dihydroxybenzamides useful as intermediates in the preparation of the corresponding alkylcarbamoyloxy derivatives. Chemical Abstracts, 74, 112752f (1971) is another disclosure of 3,5-dihydroxybenzohydroxamic acid. The compound was condensed with meta-dihydroxybenzene and formaldehyde to provide a resin. Applicants' publications relating to the activity of di- and trihydroxybenzohydroxamic acids, amides and esters as ribonucleotide reductase inhibitors with antineoplastic activity include papers appearing in Cancer Research, 39, 844 (1979), J. Med. Chem, 22, 589 (1979), Proc. Am. Assoc. Cancer Research 18, 177 (1977), 19, 63 (1978), 20, 149 (1979), Virginia Journal of Science, 29, 81 (1978), and J. Pharm. Sci., 69, 856 (1980).
3,4-Dihydroxybenzohydroxamic acid and 3,4,5-trihydroxybenzohydroxamic acid are not known nor are the corresponding phenylacetohydroxamic acids. Phenolic esters of certain hydroxybenzoic acids are also novel.
This invention provides a method of inhibiting the enzyme, ribonucleotide reductase, which comprises administering to a mammal carrying a tumor having a relatively high ribonucleotide reductase level an amount of a compound according to formula I below effective to inhibit ribonucleotide reductase ##STR1## wherein R.sup.1 is NH.sub.2, NHOH, NH(C.sub.1 -C.sub.3)alkyl, aryl-NH, N[(C.sub.1 -C.sub.3)alkyl].sub.2 or O-phenyl; m is 0, 1, 2 or 3; and n is 2, 3 or 4, except that when m is 0, n is 2 or 3 and R.sup.1 is NHOH, no OH can be ortho to the carboxyl.
Illustrative compounds useful in our novel method include the following benzohydroxamic acids, benzamides or phenyl hydroxybenzoates, compounds according to I wherein m is 0;
3,4-dihydroxybenzamide; PA0 2,3-dihydroxybenzamide; PA0 2,3,4-trihydroxybenzamide; PA0 3,4,5-trihydroxybenzamide (galloamide); PA0 2,4,5-trihydroxybenzamide; PA0 2,3,6-trihydroxybenzamide; PA0 phenyl 2,3-dihydroxybenzoate; PA0 phenyl 3,4-dihydroxybenzoate; PA0 phenyl 2,3,6-trihydroxybenzoate; PA0 N-n-propyl 2,4,6-trihydroxybenzamide; PA0 phenyl 3,4,5-trihydroxybenzoate (phenyl gallate); PA0 phenyl 2,4,6-trihydroxybenzoate; PA0 N-ethyl 3,4,5-trihydroxybenzamide. PA0 2,3,4-trihydroxybenzohydroxamic acid; PA0 3,4-dihydroxybenzohydroxamic acid; PA0 3,4,5-trihydroxybenzohydroxamic acid; PA0 3,5-dihydroxybenzohydroxamic acid; PA0 phenyl 2,3,4-trihydroxybenzoate; PA0 N-methyl 2,3,4-trihyroxybenzamide; PA0 N-ethyl 3,4,5-trihydroxybenzamide; PA0 N-n-propyl 3,4,5-trihydroxybenzamide; PA0 N-phenyl 3,4-dihydroxybenzamide, PA0 2,3,4,5-tetrahydroxybenzohydroxamic acid, PA0 2,3,4,6-tetrahydroxybenzohydroxamic acid, PA0 2,3,4,5-tetrahydroxybenzamide, PA0 phenyl 2,3,4,5-tetrahydroxybenzoate, and the like. PA0 3,4-dihydroxyphenylacetamide, PA0 .beta.-(3,4-dihydroxyphenyl)propionamide, PA0 .beta.-(3,4-dihydroxyphenyl)butyramide, PA0 .beta.-(3,4-dihydroxyphenyl)butyrohydroxamic acid, PA0 .gamma.-(3,4-dihydroxyphenyl)butyrohydroxamic acid, PA0 2,4,5-trihydroxyphenylacetohydroxamic acid, PA0 phenyl 2,3,5-trihydroxyphenylacetate, PA0 phenyl .beta.-(2,3,6-trihydroxyphenyl)propionate, PA0 phenyl .gamma.-(2,3-dihydroxyphenyl)butyrate, PA0 .beta.-(3,4-dihydroxyphenyl)propionohydroxamic acid, PA0 .gamma.-(3,4-dihydroxyphenyl)butyramide, PA0 2,3-dihydroxyphenylacetohydroxamic acid, PA0 3,4,5-trihydroxyphenylacetamide, PA0 3,4,5-trihydroxyphenylacetohydroxamic acid, PA0 N-methyl .beta.-(2,3,4-trihydroxyphenyl)propionamide, PA0 N-ethyl .gamma.-(2,3,5-trihydroxyphenyl)butyramide, PA0 phenyl .beta.-(3,4,5-trihydroxyphenyl)butyrate, PA0 .beta.-(2,3,6-trihydroxyphenyl)butyrohydroxamic, PA0 3,4-dihydroxyphenylacetohydroxamic acid, PA0 .beta.-(2,4-dihydroxyphenyl)propionamide, PA0 3,5-dihydroxyphenylacetamide, PA0 2,3,4,5-tetrahydroxyphenylacetamide, PA0 2,3,4,6-tetrahydroxyphenylpropionohydroxamic acid, PA0 phenyl 2,3,4,5-tetrahydroxyphenylacetate, and the like.
Benzohydroxamic acids, benzamides and phenyl hydroxybenzoates of phenylalkanoates (I wherein m is 1, 2 or 3) are illustrated by the following compounds:
In the above formula, when R.sup.1 is (C.sub.1 -C.sub.3) alkyl NH or [(C.sub.1 -C.sub.3)alkyl].sub.2 N, methylamino, ethylamino, dimethylamino, n-propylamino, isopropylamino, diethylamino and the like are contemplated. The term "aryl" in "aryl-NH" includes any aromatic radical such as phenyl, thienyl, pyrimidinyl and the like as well as an aryl radical substituted with one or more standard substituting groups such as halo (Cl, Br, I, F) lower alkyl (methyl, ethyl, propyl), lower alkoxy (methoxy, ethoxy), nitro, cyano etc.
A particularly useful, and therefore preferred, method of inhibiting ribonuclease reductase employs compounds of the following structure II ##STR2## wherein Z and Z' are H or OH, at least one being OH and R.sup.1 is NHOH or phenyl. Among compounds having the above structure, we specially prefer to use those compounds wherein R.sup.1 is NHOH; i.e., the benzohydroxamic acids. We also prefer to use compounds in which two hydroxyls in the phenyl ring are vicinal; i.e., compounds containing a 3,4-dihydroxyphenyl or a 3,4,5-trihydroxyphenyl group.
This invention also provides certain novel compounds; specifically, it provides compounds according to the above formula in which R.sup.1 is NHOH, compounds according to I in which R.sup.1 is O-phenyl and compounds in which m is 1, 2 or 3. In addition, in a preferred aspect of this invention, there are provided compounds in which n is 2 or 3, two of the hydroxyl groups are at the 3 to 4 carbons of the benzene ring and m is 0. A particularly preferred group included those compounds having the above structural features in which R.sup.1 is NHOH and m is 0, particularly 3,4-dihydroxybenzohydroxamic acid and 3,4,5-trihydroxybenzohydroxamic acid.
The novel compounds of this invention according to formula I above in which R is NHOH and m is 0 are prepared by reacting the corresponding ester with hydroxylamine in the presence of sodium hydroxide and sodium sulfite. The following examples illustrate the preparation of hydroxamic acids.